Project Summary Alzheimer?s disease (AD) and cerebrovascular disease account for over 80% of dementia diagnoses. Clinical, pathologic, and neuroimaging data indicate there is a synergistic relationship between cerebrovascular disease and Alzheimer?s disease. This high degree of co-morbidity implies a neurobiologic link, yet no clear molecular relationship has been established between these two common age-related impairments. In this proposal, we provide preliminary data that directly link these pathologies together and seek to identify the role that a specific stroke-activated molecular process plays in potentiating tau aggregation. Using an approach developed by the Co-PI to perform layer-specific neuronal capture and analysis after stroke, we identified that subcortical ischemic axonal injury within white matter leads to up-regulation of the microtubule-associated regulatory kinase, Mark4 in layer 5 cortical projection neurons. Mark4 is found in association with neurofibrillary tangles, phosphorylates tau at Ser262, and acts as a gateway phosphorylation event for downstream tau phosphorylation events including those that precipitate tau aggregation. These findings indicate that subcortical axonal ischemic injury primes neuronal tau phosphorylation and suggests a two-hit hypothesis that can explain the common pathologic overlap between cerebrovascular injury and AD. Here, we hypothesize that subcortical stroke primes tau aggregation through Mark4 expression and increases neuronal sensitivity to the other major pathologic hallmark of AD: extracellular b-amyloid. To prove that Mark4 acts a link between cerebrovascular injury and dementia pathologies, we will determine the effect of subcortical white matter stroke in PS19 transgenic mice that accumulate tau aggregates (Aim 1a). We will also determine the additive effect of subcortical stroke on cognitive and motor behavior in PS19 mice. In Aim 1b, we will use a peptide inhibitor discovered in bacteria to block Mark4 activity in cortical neurons and determine if we can prevent stroke-induced tau phosphorylation and aggregation in vivo (Aim 1b). In support of our hypothesis that subcortical ischemic axonal injury primes neurons for tau aggregation, we will use a novel biosensor assay developed by the Co-PI. Preliminary data using this novel assay shows that b-amyloid increases the propensity of tau to aggregate. Here, we hypothesize that Mark4 activity further primes b-amyloid-mediated tau aggregation and will test this in human biosensor cells (Aim 2a) and cultured neurons and iPSC-derived cultured neurons (Aim 2b). Together, these studies will expand our understanding of common molecular pathways in neurodegenerative disease that contribute to cognitive impairment and identify a novel therapeutic approach to target tau accumulation in mixed dementia.